Researchers in Iran have developed a protocol to detect and quantify paroxysmal nocturnal hemoglobinuria (PNH) in erythrocytes and leukocytes using multi-color flow cytometry. They described their technique, and initial findings with its use, in a recent report in the journal EJHaem.

PNH is a rare condition that can be associated with fatalities, most often resulting from thrombosis. Many diagnostic tests for the condition utilize hemolysis of red blood cells (RBCs). “Today, it is well-understood that not only RBCs but also granulocytes and monocytes are affected by PNH and the diagnosis of PNH, particularly in patients with a small population of abnormal cells, needs a highly sensitive technique,” the researchers explained in their report.

The researchers developed and standardized assays to detect PNH among RBCs and white blood cells (WBCs). They used blood samples obtained from 671 patients in the course of investigations for PNH during the years of 2014 through 2019. The researchers treated RBCs, granulocytes, and monocytes with cell type-specific monoclonal antibodies labeled with markers for detecting PNH clones, and then they performed flow cytometry for PNH clone assessment. Initially, they used a 3-color assay, but then switched to a 4-color assay for detecting and quantifying PNH clones. They also used blood samples from patients without PNH as control samples for comparisons.

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Of the 671 patients referred for assessment, 12.1% of them, or 81 patients, became diagnosed with PNH. Patients diagnosed with PNH had a mean age of 38 years (range, 11-79). Slightly more than half of the patients diagnosed with PNH were males (53.1%), while 46.9% were females. Among the 81 patients with PNH, a clinical history was available for 59 of them, with 38 patients having anemia in their histories. Neutropenia was present in 51.9% of the patients diagnosed with PNH, while anemia was present in 91.4%, and thrombocytopenia was found in 74.1%. Leukopenia was found in 51.8% of patients.

Assessments of PNH clones yielded an average total PNH clone size on RBCs of 29.58% (range, 0.34%-87.8%). Monocytes had an average PNH clone size of 58.85% (range, 1.59%-99%), and granulocytes had an average PNH clone size of 58.94% (range, 0.07%-98.44%).

Analyses of PNH clone sizes by sex showed that males and females did not have statistically significant differences in clone sizes across RBCs, granulocytes, and monocytes. There were trends of differences in PNH clone sizes between patient age groups. Patients between 25 and 45 years of age generally had larger PNH clone sizes, which differed significantly from other age groups.

“In agreement with former studies, results of the current report show that the PNH clone sizes on RBCs are significantly smaller than clones on WBCs with a p-value <0.0001 using a Mann-Whitney test,” the researchers concluded in their report. They also determined that there were correlations between PNH clone sizes and differing lineages of cells. However, they also noted that the study was limited by incomplete patient clinical histories and a small population size, so they recommended a larger study be performed.


Jahangirpour M, Vahedi A, Baghdadi H, et al. Paroxysmal nocturnal haemoglobinuria, diagnosis and haematological findings, first report from Iran, model for developing countries. EJHaem. 2022;3(2):335-340. doi:10.1002/jha2.410